Over the past decade, the unique intersection of the fields of stem cell biology and material science have produced a number of key observations about the interaction between stem cells and their surrounding niche. In addition to well-studied growth factors, specific intrinsic properties of the extracellular matrix appear to be sufficient to initiate stem cell differentiation, e.g. more rigid substrates produce myoblast- and osteoblasts-like cell types. However, these materials typically display only a single intrinsic matrix property or they lack the appropriate fibrillar structure, combination with growth factors, or spatiotemporal variations found in matrix during development. As a result, the utility of engineered tissues using these synthetic materials have been somewhat limited. Given the relative spatial and temporal complexity of definitive endoderm-derived tissues, e.g. digestive tract, liver, etc., a more prudent initial approach may be to better mimic the niche via the intrinsic matrix properties that are most germane to the production of definitive endoderm. Using physiologically-relevant combinations of growth factor signals and matrix properties, embryonic stem cells (ESCs) will be monitored for endoderm specification to determine which set of these cues or "design criteria" is most effective. We will then integrate our understanding of these criteria into two "smart," natural-synthetic hybrid biomaterials: 1) a thiolated hyaluronic acid and fibronectin matrix coupled to time-sensitive crosslinking from poly(ethylene glycol)-diacrylate to yield temporal variations of these intrinsic properties and 2) an interpenetrating polymer- fibronectin network to create spatial gradients and features having specific intrinsic matrix properties. Both materials will create dramatically more complex microenvironments compared to current biomaterials as well as better mimic the endoderm niche, which may improve ESC-to-endoderm differentiation. Novel techniques, such as force mapping spectroscopy, will aid in our characterization of these materials'biophysical and biochemical properties and will feedback into material design to create natural-synthetic hybrid substrates that are best-suited for ESCs. These insights will provide both a first set of clear-cut design criteria for the scaffolds as well as develop a process to create future therapeutic biomaterials. PUBLIC HEALTH RELEVANCE: Over 30 million Americans suffer from some form of chronic dysfunction of a definitive endoderm-derived organ, e.g. digestive tract, kidney, liver, etc., and given a lack of donor organs, the need to develop long-term organ replacement strategies is vital. Engineered tissues have been proposed as a means of dealing with this crisis, where embryonic stem cells would be grown into a tissue and subsequently implanted in the body to alleviate the dysfunction. Previous attempts, however, have had limited success in developing such tissues, likely since they do not address the intrinsic properties of the surrounding environment, which are known to direct stem cells into specific types of adult tissues.